滇重楼甾体糖基转移酶的克隆及功能表征  

Gene clone and functional identification of sterol glycosyltransferases from Paris polyphylla var.yunnanensis

作　　者：何敏 郭思远 尹艳 张驰 张夏楠[1] HE Min;GUO Si-yuan;YIN Yan;ZHANG Chi;ZHANG Xia-nan(School of Traditional Chinese Medicine,Capital Medical University,Beijing 100069,China;Institute of Acupuncture and Moxibustion,China Academy of Chinese Medical Sciences,Beijing 100700,China;Hunan Academy of Chinese Medicine,Changsha 410600,China)

机构地区：[1]首都医科大学中医药学院,北京100069 [2]中国中医科学院针灸研究所,北京100700 [3]湖南省中医药研究院,湖南长沙410600

出　　处：《中国中药杂志》2023年第14期3774-3785,共12页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金面上项目(81974515)。

摘　　要：该研究从滇重楼中克隆得到糖基转移酶基因PpUGT2,该基因ORF长度为1773 bp,编码蛋白质的氨基酸为590个。系统进化树分析显示,该糖基转移酶归为UGT80A亚家族,经国际糖基转移酶命名委员会命名为UGT80A49。构建原核表达载体pET28a-PpUGT2,通过诱导蛋白表达及蛋白提取,进行体外酶促实验,以UDP-葡萄糖为糖基供体,薯蓣皂素和偏诺皂素为底物,鉴定了其具有催化薯蓣皂素及偏诺皂素C-3羟基β糖基化的功能,为进一步研究其催化特性,对其进行了底物宽泛性研究,以UDP-葡萄糖为糖基供体,共选取了包括甾体及三萜类化合物在内的15个底物,发现其具有催化甾体类化合物睾酮C-17羟基糖基化的功能。通过同源建模及分子对接,预测了PpUGT2与底物薯蓣皂素及偏诺皂素之间相互作用的关键识别位点。并且通过对配体与多肽的氨基酸扫描,模拟突变,根据复合物的结合亲和力变化,在以底物为中心的5Å范围内,寻找到了最佳突变体,对突变体的设计具有参考意义。该研究为完整解析重楼皂苷生物合成途径以及甾体类糖基转移酶的结构研究奠定了基础。In this study,the authors cloned a glycosyltransferase gene PpUGT2 from Paris polyphylla var.yunnanensis with the ORF length of 1773 bp and encoding 590 amino acids.The phylogenetic tree revealed that PpUGT2 belonged to the UGT80A subfamily and was named as UGT80A49 by the UDP-glycosyltransferase(UGT)Nomenclature Committee.The expression vector pET28a-PpUGT2 was constructed,and enzyme catalytic reaction in vitro was conducted via inducing protein expression and extraction.With UDP-glucose as sugar donor and diosgenin and pennogenin as substrates,the protein was found with the ability to catalyze the C-3 hydroxylβ-glycosylation of diosgenin and pennogenin.To further explore its catalytic characteristic,15 substrates including steroids and triterpenes were selected and PpUGT2 showed its activity towards the C-17 position of sterol testosterone with UDP-glucose as sugar donor.Homology modelling and molecule docking of PpUGT2 with substrates predicted the key residues interacting with ligands.The re-levant residues of PpUGT2-ligand binding model were scanned to calculate the corresponding mutants,and the optimized mutants were obtained according to the changes in binding affinity of the ligand with protein and the surrounding residues within 5.0Åof ligands,which had reference value for design of the mutants.This study laid a foundation for further exploring the biosynthetic pathway of polyphyllin as well as the structure of sterol glycosyltransferases.

关 键 词：重楼皂苷 甾体糖基转移酶 生物合成途径 

分 类 号：S567.239[农业科学—中草药栽培]